Sensitizing protective tumor microenvironments to antibody-mediated therapy.

Therapy-resistant microenvironments represent a major barrier toward effective elimination of disseminated malignancies. Here, we show that select microenvironments can underlie resistance to antibody-based therapy. Using a humanized model of treatment refractory B cell leukemia, we find that infiltration of leukemia cells into the bone marrow rewires the tumor microenvironment to inhibit engulfment of antibody-targeted tumor cells. Resistance to macrophage-mediated killing can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, the nitrogen mustard cyclophosphamide induces an acute secretory activating phenotype (ASAP), releasing CCL4, IL8, VEGF, and TNFα from treated tumor cells. These factors induce macrophage infiltration and phagocytic activity in the bone marrow. Thus, the acute induction of stress-related cytokines can effectively target cancer cells for removal by the innate immune system. This synergistic chemoimmunotherapeutic regimen represents a potent strategy for using conventional anticancer agents to alter the tumor microenvironment and promote the efficacy of targeted therapeutics.

Densely interconnected transcriptional circuits control cell states in human hematopoiesis.

Though many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly coexpressed genes, some of which are restricted to a single lineage but most of which are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.

Clinical Presentation and Bacteriology of Eyebrow Infections: The Massachusetts Eye and Ear Infirmary Experience (2008-2015).

PURPOSE: This study retrospectively reviews preseptal cellulitis and abscesses involving the eyebrow to elucidate the bacteriology and potential causative factors. METHODS: A retrospective chart review was conducted to identify patients who had been diagnosed with preseptal cellulitis or abscess involving the eyebrow at the Massachusetts Eye and Ear Infirmary between 2008 and 2015. Demographic, clinical, and microbiological data were collected. RESULTS: Eighty patients with eyebrow infections were identified, of whom 49 (61.3%) were female and 31 (38.7%) were male. The median age was 37 years (range 14-67 years). Eyebrow abscess was present in 54 cases (67.5%), while 26 cases (32.5%) were limited to preseptal cellulitis without abscess formation. Methicillin-resistant Staphylococcus aureus was found in 20 abscesses (39.2% of culture results), and methicillin-sensitive S. aureus was found in 12 abscesses (23.5% of culture results). Coagulase-negative staphylococci were present in 7 eyebrow abscesses (13.7% of culture results). Clinical history was remarkable for eyebrow hair removal (tweezing, waxing, threading, or shaving) in 17 cases (21.3%), manipulation of acne lesions ("popping," "picking," or "squeezing") in 6 cases (7.5%), and both brow hair removal and acne manipulation in 1 case (1.3%). CONCLUSIONS: There is a high incidence of methicillin-resistant Staphylococcus aureus in the bacteriology of eyebrow infections. Empirical antibiotic coverage for methicillin-resistant Staphylococcus aureus should be strongly considered in any patient with an eyebrow area abscess or preseptal cellulitis. Individuals who practice cosmetic eyebrow grooming should be encouraged to consider hygiene practices, which could reduce the risk of infection.

Intermittent Horner syndrome in a pediatric patient.

Intermittent Horner syndrome is uncommon in both the adult and pediatric population. We describe a case of a pediatric patient with an intermittent Horner syndrome. Infrared photography and videography were used to help establish the diagnosis.

Urrets-Zavalia syndrome following placement of scleral-sutured intraocular lens.

Purpose: To report a novel case of Urrets-Zavalia syndrome (UZS). Observation: A 59-year-old man underwent removal of a dislocated intraocular lens and placement of a scleral-sutured intraocular lens. After surgery, the pupil in the operative eye was dilated, fixed, and unresponsive to constricting drops. Conclusion: This case expands the known etiology of UZS. Possible preventative measures may include pre-operative screening for plateau iris and intra-operative use of iris hooks instead of pharmacological dilation.

Antigen-bearing dendritic cells regulate the diverse pattern of memory CD8 T-cell development in different tissues.

Memory T cells of the effector type (T(EM)) account for the characteristic rapidity of memory T-cell responses, whereas memory T cells of the central type (T(CM)) account for long-lasting, vigorously proliferating memory T-cell responses. How antigen-stimulated (primed) T cells develop into different memory T-cell subsets with diverse tissue distributions is largely unknown. Here we show that after respiratory tract infection of mice with influenza virus, viral antigen associated with dendritic cells (DCs) was abundant in lung-draining lymph nodes (DLN) and the spleen for more than a week but was scant and transient in nondraining lymph nodes (NDLN). Correspondingly, activated CD8 T cells proliferated extensively in DLN and the spleen but minimally in NDLN. Strikingly, however, although most persisting CD8 T cells in DLN and spleen exhibited the T(EM) phenotype, those persisting in NDLN exhibited the T(CM) phenotype. Reducing antigen exposure by depleting DCs at the peak of primary T-cell responses enhanced the development of T(CM), whereas subjecting primed CD8 T cells from NDLN to additional antigen stimulation inhibited T(CM) development. These findings demonstrate that differences in persistence of antigen-bearing DCs in various tissues regulate the tissue-specific pattern of memory CD8 T-cell development. The findings have significant implications for design of vaccines and immunization strategies.

Age-Related Differences in Ocular Features of a Naturalistic Free-Ranging Population of Rhesus Macaques.

Purpose: Rhesus macaques (Macaca mulatta) are the premier nonhuman primate model for studying human health and disease. We investigated if age was associated with clinically relevant ocular features in a large cohort of free-ranging rhesus macaques from Cayo Santiago, Puerto Rico. Methods: We evaluated 120 rhesus macaques (73 males, 47 females) from 0 to 29 years old (mean ± SD: 12.6 ± 6.4) from September to December 2021. The ophthalmic evaluation included intraocular pressure (IOP) assessment, corneal pachymetry, biomicroscopy, A-scan biometry, automated refraction, and fundus photography after pupil dilation. The associations of age with the outcomes were investigated through multilevel mixed-effects models adjusted for sex and weight. Results: On average, IOP, pachymetry, axial length, and automated refraction spherical equivalent were 18.37 ± 4.68 mmHg, 474.43 ± 32.21 µm, 19.49 ± 1.24 mm, and 0.30 ± 1.70 diopters (D), respectively. Age was significantly associated with pachymetry (ß coefficient = -1.20; 95% confidence interval [CI], -2.27 to -0.14; P = 0.026), axial length (ß coefficient = 0.03; 95% CI, 0.01 to 0.05; P = 0.002), and spherical equivalent (ß coefficient = -0.12; 95% CI, -0.22 to -0.02; P = 0.015). No association was detected between age and IOP. The prevalence of cataracts in either eye was 10.83% (95% CI, 6.34-17.89) and was significantly associated with age (odds ratio [OR] = 1.20; 95% CI, 1.06-1.36; P = 0.004). Retinal drusen in either eye was observed in 15.00% (95% CI, 9.60-22.68) of animals, which was also significantly associated with age (OR = 1.14; 95% CI, 1.02-1.27; P = 0.020). Conclusions: Rhesus macaques exhibit age-related ocular associations similar to those observed in human aging, including decreased corneal thickness, increased axial length, myopic shift, and higher prevalence of cataract and retinal drusen.

A NOVEL APPROACH TO SCLERAL FIXATION OF POSTERIOR CHAMBER INTRAOCULAR LENSES AND CAPSULAR TENSION RINGS AND SEGMENTS IN DEEP-SET EYES.

PURPOSE: To demonstrate a novel approach to scleral fixation of posterior chamber intraocular lenses and capsular tension rings and segments in deep-set eyes using the Finesse FlexLoop (Alcon Laboratories). METHODS: The technique described herein, based on previous approaches to scleral fixation of posterior chamber intraocular lenses, uniquely employs the FlexLoop to "lasso" Gore-Tex sutures that have already been threaded through the eyelets of a CZ70BD (Alcon Laboratories) IOL and externalize them. RESULTS: All patients who underwent surgery with this technique experienced visual improvement. The only complication was of mild hyphema in the patient who had a capsular tension segment placed, which resolved with medical therapy. CONCLUSION: The advantages of this procedure include a smaller diameter instrument (FlexLoop) as compared to the 25-gauge forceps typically employed, an easier to perform surgical maneuver that alleviates the need for both precise placement and constant tension to be exerted by the surgeon to grasp the sutures, as well as an instrument that can function when bent up to 45° to help accommodate deep-set eyes requiring this procedure.

SEROUS MACULAR DETACHMENT ASSOCIATED WITH WALDENSTROM MACROGLOBULINEMIA MANAGED WITH IBRUTINIB: A CASE REPORT AND NEW INSIGHTS INTO PATHOGENESIS.

PURPOSE: To report a case of serous macular detachment in a patient with Waldenstrom macroglobulinemia treated with ibrutinib. METHODS: The patient underwent a complete ophthalmic examination and imaging at presentation and at follow-up visits up to 13 months. RESULTS: At presentation, there were serous macular detachments bilaterally with no dye leakage on fluorescein angiography or vasculature abnormalities on optical coherence tomography angiography. After treatment with ibrutinib, there was near resolution of the patient's retinopathy with an improvement in vision at 13 months' follow-up. CONCLUSION: Serous macular detachments in Waldenstrom macroglobulinemia-associated retinopathy may be due to the disruption of the retinal pigment epithelium pump mechanism by hyperglobulinemia. The favorable course of this patient, treated with the novel tyrosine kinase inhibitor ibrutinib, suggests this may be the preferred treatment for Waldenstrom macroglobulinemia patients with associated retinopathy.

The Proteome of Preretinal Tissue in Proliferative Vitreoretinopathy.

BACKGROUND AND OBJECTIVE: Proliferative vitreoretinopathy (PVR) is the leading cause of retinal detachment repair failure. However, the molecular pathogenesis remains incompletely understood. Determining the proteome of PVR will help to identify novel therapeutic targets. MATERIALS AND METHODS: Preretinal tissue samples, delaminated during surgery from six PVR cases and one idiopathic epiretinal membrane (ERM) were analyzed by mass spectrometry. Tandem mass spectra were extracted using the UniProt database, generating a list of 896 proteins, which were subjected to pathway set and fold-change (ERM vs PVR) analyses. RESULTS: Two pathways were enriched in PVR: extracellular matrix (ECM) organization and extracellular structure organization. A fold-change analysis comparing mean total spectral counts from PVR to an ERM control identified fibronectin, the ECM glycoprotein, as the protein most significantly elevated in PVR compared to ERM. CONCLUSION: These data identify pathwayskey to PVR progression, including thoseinvolved in cell-mediated ECM assembly and thus tractional force generation at the cellular level. [Ophthalmic Surg Lasers Imaging Retina. 2021;52:S5-S12.].

Diffuse Uveal Melanocytic Proliferation With Primary Vitreoretinal Lymphoma.

Importance: Bilateral diffuse uveal melanocytic proliferation is a rare sign of several systemic malignant neoplasms. Observations: A patient presenting with uveal melanocytic proliferation underwent a detailed physical examination and extensive imaging. No systemic malignant neoplasm was found. Chorioretinal biopsy was performed, and its immunohistochemical results revealed the presence of primary vitreoretinal lymphoma. Conclusions and Relevance: This patient's results suggest that diffuse uveal melanocytic proliferation may be associated not just with systemic malignant disease, but also with primary intraocular tumors, in this case a primary vitreoretinal lymphoma.

Phage Immunoprecipitation Sequencing of Autoantigens in Autoimmune Retinopathy.

PURPOSE: To identify autoantigens in autoimmune retinopathy patients by phage immunoprecipitation sequencing (PhIP-Seq), a new technique for autoantigen discovery. METHODS: PhIP-Seq was used to sequence putative autoantibodies in plasma from 11 patients with autoimmune retinopathy and eight controls. We compared the autoantibodies' molecular weights with those of proteins detected by Western blot. RESULTS: Several autoantigens were found in cases and not detected in the controls. Autoantigens RTN3, PRPF6, TRPC6, and B3GNT8, four proteins expressed in the retina, were detected in plasma as autoantibodies from one patient each and no controls. Only one patient had an autoantibody, B3GNT8 (43.4 kDa), within a similar weight range as that detected by antiretinal antibody Western blot (42 kDa). Autoantibody POLR3A, which has a well-characterized role in scleroderma, was detected in two cases and no controls. CONCLUSION: PhIP-Seq detected autoantigens that are expressed in the retina as well as scleroderma-related autoantigens in autoimmune retinopathy patients.

The DEP domain determines subcellular targeting of the GTPase activating protein RGS9 in vivo.

DEP (for Disheveled, EGL-10, Pleckstrin) homology domains are present in numerous signaling proteins, including many in the nervous system, but their function remains mostly elusive. We report that the DEP domain of a photoreceptor-specific signaling protein, RGS9 (for regulator of G-protein signaling 9), plays an essential role in RGS9 delivery to the intracellular compartment of its functioning, the rod outer segment. We generated a transgenic mouse in which RGS9 was replaced by its mutant lacking the DEP domain. We then used a combination of the quantitative technique of serial tangential sectioning-Western blotting with electrophysiological recordings to demonstrate that mutant RGS9 is expressed in rods in the normal amount but is completely excluded from the outer segments. The delivery of RGS9 to rod outer segments is likely to be mediated by the DEP domain interaction with a transmembrane protein, R9AP (for RGS9 anchoring protein), known to anchor RGS9 on the surface of photoreceptor membranes and to potentiate RGS9 catalytic activity. We show that both of these functions are also abolished as the result of the DEP domain deletion. These findings indicate that a novel function of the DEP domain is to target a signaling protein to a specific compartment of a highly polarized neuron. Interestingly, sequence analysis of R9AP reveals the presence of a conserved R-SNARE (for soluble N-ethylmaleimide-sensitive factor attachment protein receptor) motif and a predicted overall structural homology with SNARE proteins involved in vesicular trafficking and fusion. This presents the possibility that DEP domains might serve to target various DEP-containing proteins to the sites of their intracellular action via interactions with the members of extended SNARE protein family.

Homeostasis of T cell diversity.

T cell homeostasis commonly refers to the maintenance of relatively stable T cell numbers in the peripheral lymphoid organs. Among the large numbers of T cells in the periphery, T cells exhibit structural diversity, i.e., the expression of a diverse repertoire of T cell receptors (TCRs), and functional diversity, i.e., the presence of T cells at naive, effector, and memory developmental stages. Although the homeostasis of T cell numbers has been extensively studied, investigation of the mechanisms underlying the maintenance of structural and functional diversity of T cells is still at an early stage. The fundamental feature throughout T cell development is the interaction between the TCR and either self or foreign peptides in association with MHC molecules. In this review, we present evidence showing that homeostasis of T cell number and diversity is mediated through competition for limiting resources. The number of T cells is maintained through competition for limiting cytokines, whereas the diversity of T cells is maintained by competition for self-peptide-MHC complexes. In other words, diversity of the self-peptide repertoire limits the structural (TCR) diversity of a T cell population. We speculate that cognate low affinity self-peptides, acting as weak agonists and antagonists, regulate the homeostasis of T cell diversity whereas non-cognate or null peptides which are extremely abundant for any given TCR, may contribute to the homeostasis of T cell number by providing survival signals. Moreover, self-peptides and cytokines may form specialized niches for the regulation of T cell homeostasis.

Rapid generation of human B-cell lymphomas via combined expression of Myc and Bcl2 and their use as a preclinical model for biological therapies.

Although numerous mouse models of B-cell malignancy have been developed via the enforced expression of defined oncogenic lesions, the feasibility of generating lineage-defined human B-cell malignancies using mice reconstituted with modified human hematopoietic stem cells (HSCs) remains unclear. In fact, whether human cells can be transformed as readily as murine cells by simple oncogene combinations is a subject of considerable debate. Here, we describe the development of humanized mouse model of MYC/BCL2-driven 'double-hit' lymphoma. By engrafting human HSCs transduced with the oncogene combination into immunodeficient mice, we generate a fatal B malignancy with complete penetrance. This humanized-MYC/BCL2-model (hMB) accurately recapitulates the histopathological and clinical aspects of steroid-, chemotherapy- and rituximab-resistant human 'double-hit' lymphomas that involve the MYC and BCL2 loci. Notably, this model can serve as a platform for the evaluation of antibody-based therapeutics. As a proof of principle, we used this model to show that the anti-CD52 antibody alemtuzumab effectively eliminates lymphoma cells from the spleen, liver and peripheral blood, but not from the brain. The hMB humanized mouse model underscores the synergy of MYC and BCL2 in 'double-hit' lymphomas in human patients. Additionally, our findings highlight the utility of humanized mouse models in interrogating therapeutic approaches, particularly human-specific monoclonal antibodies.

Human CD34+ CD133+ hematopoietic stem cells cultured with growth factors including Angptl5 efficiently engraft adult NOD-SCID Il2rγ-/- (NSG) mice.

Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+) CD133(+) cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/-) (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+) CD133(+) fraction of expanded cells and that CD34(+) CD133(+) cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.

Mesenchymal stem cells secreting angiopoietin-like-5 support efficient expansion of human hematopoietic stem cells without compromising their repopulating potential.

Clinical and preclinical applications of human hematopoietic stem cells (HSCs) are often limited by scarcity of cells. Expanding human HSCs to increase their numbers while maintaining their stem cell properties has therefore become an important area of research. Here, we report a robust HSC coculture system wherein cord blood CD34(+) CD133(+) cells were cocultured with mesenchymal stem cells engineered to express angiopoietin-like-5 in a defined medium. After 11 days of culture, SCID repopulating cells were expanded ~60-fold by limiting dilution assay in NOD-scid Il2rg(-/-) (NSG) mice. The cultured CD34(+) CD133(+) cells had similar engraftment potential to uncultured CD34(+) CD133(+) cells in competitive repopulation assays and were capable of efficient secondary reconstitution. Further, the expanded cells supported a robust multilineage reconstitution of human blood cells in NSG recipient mice, including a more efficient T-cell reconstitution. These results demonstrate that the expanded CD34(+) CD133(+) cells maintain both short-term and long-term HSC activities. To our knowledge, this ~60-fold expansion of SCID repopulating cells is the best expansion of human HSCs reported to date. Further development of this coculture method for expanding human HSCs for clinical and preclinical applications is therefore warranted.